Ostrinia furnacalis (Guenée), is the most destructive lepidopteran insect pest of corn (Zea mays L.) in China. Pathogenic microorganisms play an important role in the population control of the Asian corn borer. Although microorganisms can cause the death of O. furnacalis, an immune response also occurs as an attempt to fight off and eliminate invading pathogens. If the molecular mechanism of interaction between O. furnacalis and pathogenic bacteria is clarified, the lethal effect of pathogenic microorganisms can be better exerted by inhibiting the natural immune response of O. furnacalis. As an important member of the pattern-recognition receptor family, peptidoglycan recognition protein (PGRP) plays a key role in the insect innate immune response. In this study, we cloned two PGRP genes from O. furnacalis and analyzed their spatiotemporal expression. In combination with bacterial induction experiments, we revealed the immune signal recognition pathway involved in the two proteins. The results of this study deepen the understanding of the natural immune response of O. furnacalis and provide new ideas for better utilization of pathogenic microorganisms in biological control of the Asian corn borer.The Asian corn borer, OfPGRP-A and OfPGRP-B, from the Asian corn borer, Ostrinia furnacalis. These two genes comprise open reading frames of 658 and 759 bp, encoding proteins of 192 and 218 amino acids, respectively. qPCR showed that OfPGRP-A and OfPGRP-B are prominently expressed in the midgut of O. furnacalis fourth instar larvae. After inoculation with Staphylococcus aureus and Bacillus thuringiensis, the expression of OfPGRP-A was significantly upregulated, whereas the expression of OfPGRP-B was enhanced after inoculation with Escherichia coli. This suggests that OfPGRP-A mainly recognizes Gram-positive bacteria and may participate in the Toll signaling pathways, while OfPGRP-B identifies Gram-negative bacteria and may participate in Imd signaling pathways. Our results provide insights into the roles of PGRPs in O. furnacalis immune function and a foundation for using pathogens for the biological control of O. furnacalis.Peptidoglycan recognition proteins (PGRPs) are important components of insect immune systems, in which they play key roles. We cloned and sequenced two full-length PGRP, named  Innate immunity is the first defense in insects and mammals against bacterial, fungal, viral, and parasitic infections. Identifying invading pathogens is key to triggering the host immune responses. Peptidoglycan recognition protein (PGRP), a member of the pattern-recognition receptor family, can recognize pathogen-associated molecular patterns present on the pathogen surface. After recognition, the Toll signaling pathway, Imd pathway, JAK–STAT pathway, reactive oxygen metabolism, and a melanization reaction are selectively activated to eliminate pathogens ,2,3.Bombyx mori, and then in mammals (humans and mice) [N-acetylmuramic acid-L-alanine amidase [Drosophila melanogaster, although PGRP-S is secreted in different parts of the body, it is mostly expressed in immune organs, particularly after microbial infection. PGRP-SB1, SC2, and SD are highly expressed in the fat body, PGRP-SA is expressed in the epithelial tissue, and PGRP-SC is specifically expressed in viscera [PGRP was first found in the blood of nd mice) . More thnd mice) . These p amidase . PGRPs h amidase . PGRPs c amidase ,9. Insec viscera .D. melanogaster bind with Gram-negative bacteria binding protein 1 to activate the Toll signaling pathway [D. melanogaster can recognize DAP-PGN on the cell wall of Gram-negative bacteria, and the PGRP functional area outside the cell membrane binds to PGN to activate the Imd signaling pathway [D. melanogaster and B. mori recognize bacteria, initiate immune responses, and participate in signaling pathways, whereas relatively few studies have focused on other lepidopterans.PGRPs play important roles in identifying invading pathogens and regulating the innate immune responses, and they can directly kill invading pathogens. PGRPs can recognize pathogenic microorganisms and activate the Toll signaling pathway and Imd pathway, which induces the synthesis of antimicrobial peptides. These two pathways are activated by Lys-type and DAP-type peptidoglycans (PGNs), respectively . Dm PGRP pathway . Dm PGRP pathway . DiffereOstrinia furnacalis (Guenée), is the most destructive lepidopteran insect pest of corn (Zea mays L.) in China [O. furnacalis can bore into stalks and ear shanks, feeding on whorl leaves and young kernels, thus resulting in yield losses. The use of entomopathogens for controlling O. furnacalis has recently received increasing attention [The Asian corn borer, in China . It is win China . The larttention ,17. HoweO. furnacalis. After sequence alignment, these genes were named OfPGRP-A (Gene ID:ON152884) and OfPGRP-B (Gene ID: ON152885). In this study, we conducted cloning and bioinformatics analysis of these two genes. Based on the tissue expression patterns of OfPGRP-A and OfPGRP-B after bacterial inoculation, we explored the role of the two genes in the innate immune response. We predicted the functions of OfPGRP-A and OfPGRP-B proteins and their involvement in immune signal pathways. This study provides a theoretical basis for using microbial pesticides to control Asian corn borer and for developing more efficient and environmentally friendly pesticides.Initially, two PGRPs were identified by analyzing full-length transcriptome sequences of pathogen-induced O. furnacalis larvae were reared at 26 ± 1°C, RH = 80% ± 5%, and a photoperiod of 16 L: 8 D. The larvae were fed on a semi-artificial diet as described by Zhou [ by Zhou . The larStaphylococcusaureus and Bacillus thuringiensis and the Gram-negative bacterium Escherichia coli. These three bacterial strains were purchased from the Institute of Microbiology, Chinese Academy of Sciences . The bacteria were activated and then cultured at 37 °C and 200 rpm until the OD600 reached around 1. After centrifugation at 5000× g for 5 min at 4 °C, the supernatant was removed. The bacteria were collected, washed three times with sterile phosphate-buffered saline (pH 6.4), and resuspended to the required bacterial concentration. After autoclaving at 121 °C for 20 min, they were kept at −20 °C until use.The bacteria used in this experiment were the Gram-positive bacteria O. furnacalis. First, 1% agarose gel electrophoresis was used to detect the quality of the total RNA, then a Nanodrop 2000  was used to detect the concentration and purity of the total RNA, and an Agilent 2100 bioanalyzer  was used to determine the RNA integrity to ensure the quality of the RNA samples. Finally, high-quality RNA samples were reverse transcribed to synthesize cDNA using a TransScript One-step gDNA Removal and cDNA Synthesis SuperMix kit .The TRIzol method was used to extract total RNA from O. furnacalis, two PGRP gene sequences were obtained. Based on sequence alignment and phylogenetic tree analysis, these genes were named OfPGRP-A and OfPGRP-B. Based on the cDNA sequences of OfPGRP-A and OfPGRP-B, primers were designed using Primer Premier 5.0 software  and DNAMAN5.0 , respectively  into O. furnacalis was used as a template to amplify the full-length sequence of two PGRPs. The 50-μL reaction system contained 1 µL cDNA template, 1 µL each of upstream and downstream primers (10 mmol/L), 25 µL Taq PCR Master Mixblue dye (2x), and 22 µL ddH2O. The PCR conditions were as follows: 94 °C for 3 min, 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min for 30 cycles, followed by 72 °C for 10 min. The PCR product was detected by 1% agarose gel electrophoresis. The target band was recovered using an AxyGen DNA gel recovery kit  and ligated into the pGEM-T Easy vector . The ligation product was transformed into E. coli DH5α competent cells, and positive clones were screened using PCR with M13 universal primers. Finally, the samples were sent for sequencing to Sangon Biotech Co., Ltd. .According to the transcriptome sequencing results of ectively . The cDNO. furnacalis PGRP genes to obtain complete cDNA sequences. ExPASy  was used to predict the molecular weight and isoelectric point of the proteins. SMART  was used to analyze functional domains in the protein sequences. The online software SignalP 5.0  was used to analyze signal peptides. TMHMM Server 2.0  was used to analyze the transmembrane domain. We downloaded the PGRP sequences of other insects from NCBI, used Clustal X2.0 for homology alignment analysis, and used Jalview software 2.10 for multiple sequence alignment editing. We also used MEGA 7.0 software for neighbor-joining analysis to construct a phylogenetic tree, with bootstrapping 1000 times for testing.We spliced and evaluated the sequenced O. furnacalis larvae from the 1st–5th instars and dissected 4th instars to collect samples from different tissues . Sixty 4th instar O. furnacalis larvae were randomly selected, their stomachs and feet were disinfected with 70% alcohol, and they were placed on ice for 5 min for freezing and anesthesia. Next, we sterilized the larvae’s gastropod using a sterilized insect needle, gently squeezed the gastropod to drop the hemolymph on parafilm, and this sample was placed in a 1.5 mL centrifuge tube. The epidermis was dissected, and the midgut, fat body, and body wall were separately collected. The hemolymph was centrifuged at 4 °C and 12,000× g for 30 min, and the supernatant was collected. All tissue samples were stored at −80 °C until use.We collected OfPGRP-A and OfPGRP-B in different stages and different tissues of the larvae. qPCR reaction consists of a system (20 μL): 1 μL cDNA template, 1 μL each upstream and downstream primers (10 mmol/L), 1 μL ROX Reference Dye II, 10 μL TB Green Premix Ex Taq, and 6 μL ddH2O. The program was set according to the RR420A manual as follows: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 34 s for 40 cycles, followed by 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Ostrinia furnacalis RPL-18 was used as an internal reference gene [Primer Premier 5.0 software was used to design quantitative primers . A Takarnce gene .O. furnacalis of the same size were selected and divided into a control group, phosphate-buffered saline injection group, S. aureus injection group, E. coli injection group, and B. thuringiensis injection group. Thirty larvae were injected for each treatment as described by Sun and Bai [6 cells/mL); this solution was injected into the larval gastropod. After injection, the larvae surfaces were disinfected with 70% alcohol. The larvae were then reared at 26 ± 1 °C, RH = 80 ± 5%, and a photoperiod of 16 L: 8 D.Fourth instar larvae of  and Bai . A microFive larvae were collected for dissection at 2, 4, 8, 12, 24, and 48 h after injecting the bacteria, and different tissue samples were collected for quantitative analysis. Three replicates were used for each treatment. The collection method and qPCR program were the same as those described in OfPGRP-A and OfPGRP-B in O. furnacalis at different instars, in different tissues, and after induction with different bacteria were calculated using the 2−∆∆Ct method [The relative expression levels of t method . DiffereO. furnacalis OfPGRP-A and OfPGRP-B were obtained by PCR amplification. The complete cDNA of OfPGRP-A consisted of 658 bp and encoded a protein of 192 amino acids , Ami2 domain (amino acids 33–170), and a N-terminal signal peptide comprising 20 amino acids. The open reading frame of OfPGRP-B was 759 bp and encoded a protein of 218 amino acids . The expression level of OfPGRP-A in 4th instar larvae was 55-fold higher than that in 1st instar larvae, and OfPGRP-B showed the lowest expression level in 1st instar larvae, with the expression being almost undetectable , followed by expression in the fat body. Expression was lowest in the hemolymph; expression in the midgut was nearly 140-fold higher than that in the hemolymph. OfPGRP-B was also highly expressed in the midgut at significantly higher levels than in the epidermis and hemolymph, indicating that the expression of OfPGRP is tissue-specific ), after which the expression gradually decreased. OfPGRP-A was also significantly upregulated at multiple time points after S. aureus induction. In the hemolymph, OfPGRP-A was significantly activated by S. aureus. The expression levels at 2, 4, 8, and 12 h after induction were significantly higher than those in the control and after other bacterial treatments. At 8 h after treatment, the expression of OfPGRP-A reached the highest level . In the fat body, OfPGRP-A expression was upregulated at all time points. At 2 and 4 h after S. aureus injection, the expression level was significantly higher than in the other groups (p < 0.05), after which the expression level decreased. The expression level of OfPGRP-A following B. thuringiensis induction reached the highest level after 12 h. After induction by E. coli, the expression level of OfPGRP-A was upregulated, although the increase was not as significant as that observed with the other two bacteria. In the midgut, the expression of OfPGRP-A reached the highest level at 12 h after each of the three bacterium injections, with the expression being significantly higher than that in the control (p < 0.05). The most significant increase in OfPGRP-A expression was induced by S. aureus. Some PGRPs act as pattern recognition receptors that recognize and bind to foreign pathogens and then activate the Toll and Imd pathways. Lysine-type PGN mainly activates the Toll pathway, while the DAP-type PGN activates the Imd pathway [OfPGRP-A expression was significantly increased after induction by the Gram-positive bacteria S. aureus and B. thuringiensis. This gene may be involved in activating the Toll pathway.Inoculation of the three bacteria caused changes in the expression level of s larvae . In the  pathway ,23. AccoOfPGRP-B gene in O. furnacalis larvae was higher than that in the control group and other bacterial treatment groups at various time points and in different tissues after E. coli injection , after which it decreased. In the hemolymph, after E. coli induction, the expression of OfPGRP-B increased, reaching the highest expression level at 8 h, and then decreased. Compared with S. aureus and B. thuringiensis, there is a significant difference in expression (p < 0.05). OfPGRP-B expression was significantly upregulated in the fat body after E. coli induction compared with that in the control group at all time points (p < 0.05). The fold-increase was particularly significant in the first eight hours. S. aureus and B. thuringiensis could also increase OfPGRP-B expression, but to lower levels than that induced by E. coli injection. In the midgut, after injection of the three bacteria, the induction effect of E. coli was the most significant, followed by S. aureus. Compared with the control, the expression of OfPGRP-B significantly differed at 4, 8, and 12 h after induction, reaching the highest level at 8 h. According to these results, the expression of OfPGRP-B increased significantly after induction by the Gram-negative bacteria E. coli. This gene may activate the Imd pathway.The expression level of the njection . In the D. melanogaster and B. mori. Two PGRP gene sequences (OfPGRP-A and OfPGRP-B) were screened from the sequence data of the transcriptome of O. furnacalis larvae by sequence comparison and evolutionary analysis. In this study, the full-length cDNA sequences of OfPGRP-A and OfPGRP-B were successfully cloned, the sequence characteristics of the genes were analyzed using bioinformatics software, and their spatiotemporal and pathogen-induced expression patterns were detected using qPCR.Insects possess innate immunity against invading microbial pathogens. PGRPs in insects play important roles in recognizing bacterial infections. These proteins can specifically recognize PGNs on bacterial surfaces, which activate the Toll and Imd pathways as well as downstream immune responses ,12. In rDrosophila melanogaster, of which seven  were type PGRP-S and contained signal peptides but no transmembrane domains. The other six were PGRP-L, which are divided into two categories: those in one category contain signal peptides and transmembrane domains, whereas members of the other contain no signal peptides nor transmembrane domains [OfPGRP-A and OfPGRP-B have conserved PGRP domains. OfPGRP-A has a signal peptide but no transmembrane domain, whereas OfPGRP-B has neither a transmembrane domain nor a signal peptide. We predicted that both genes belong to the short form of PGRP, which is a secreted extracellular protein. For most PGRPs that have amidase activity, this activity is contained in the H–Y–H–T–C structure [OfPGRP-A sequence, tyrosine is mutated to serine and cysteine is mutated to serine, suggesting that OfPGRP-A lacks amidase activity. OfPGRP-B retains five key amidase active sites of H–Y–H–T–C. It also has arginine residues in the DAP-type PGN recognition site, indicating that this gene has amidase activity. OfPGRP-B may be involved in recognizing Gram-negative bacteria, leading to activation of the Imd signaling pathway. OfPGRP-A and OfPGRP-B clustered with the PGRP genes of P. xylostella and M. sexta, respectively, indicating that PGRP has relatively conserved evolutionary characteristics in lepidopterans.Insect PGRP can be divided into short PGRP-S and long PGRP-L according to the molecular size and characteristic structure. For example, 13 PGRPs were found in  domains . Sequenctructure . SequencBdPGRP-LCa and BdPGRP-LCb of Bactrocera dorsalis increase from the mature larva to early pupal stage and from the late pupal to early adult stage. Both genes are highly expressed in the larval midgut and fat bodies but lower in the gonads. BdPGRP-SA exhibits the highest expression in adults, lowest expression in the egg and early larval stages, and high expression in the fat body and midgut [PxPGRP-S1 shows the highest expression in the 4th instar larval stage and pupal stage of Plutellaxylostella; lowest expression in the egg, 2nd instar, and adult stages; and highest expression in the fat body among tissues [OfPGRP-A and OfPGRP-B showed the highest expression in 4th instar larvae. Before the 4th instar, the expression level gradually increased with age. As the larvae grew, immune function gradually enhanced, and the ability to resist pathogens was strongest in the 4th instar larvae. The midgut is an essential immune response center in the intestine, whereas the fat body synthesizes, stores, detoxifies, and metabolizes toxic substances. Both the midgut and fat body are important immune organs that defend against pathogens [OfPGRP-A and OfPGRP-B were significantly higher in the midgut than in other tissues. After inoculation by different bacteria, OfPGRP-A and OfPGRP-B expression changed significantly in each tissue to different extents. Thus, OfPGRP expression may differ between tissues.PGRPs show significantly different expression levels at various developmental stages as well as tissue-specific expression. The expression levels of d midgut ,27. PxPG tissues . In thisathogens . In thisDrosophila, PGRP-SA can bind to lysine-type PGN and activate the Toll pathway [OfPGRP-A expression increased significantly after injection of S. aureus and B. thuringiensis, and OfPGRP-B expression increased significantly after injection of E. coli. S. aureus and B. thuringiensis are Gram-positive bacteria with lysine-type PGN, whereas E. coli are Gram-negative bacteria with DAP-type PGN. The change in PGRP gene expression after injection with different bacteria indicates that PGRP regulates different signaling pathways. Combined with previous analysis of the gene characteristics, OfPGRP-A may be involved in activation of the Toll pathway, and OfPGRP-B may be involved in activation of the Imd pathway. In further studies, we will obtain the recombinant proteins of OfPGRP-A and OfPGRP-B using a prokaryotic expression system and perform in vitro experiments to explore the functions of OfPGRP-A and OfPGRP-B proteins, thus determining the possible immune signal pathways in which they participate. This study lays a foundation for further research on the function of PGRP in O. furnacalis and will help facilitate the use of entomopathogens for control of this insect pest.Insect PGRPs can recognize PGN in the bacterial cell wall and activate the Toll and Imd pathways . PGRP ac pathway , whereas pathway . In this


Here, we used a genome-wide de novo approach to investigate preferentially located motifs (PLMs) in the proximal cis-regulatory landscape of Arabidopsis thaliana and Zea mays. We report three groups of PLMs in both the 5’- and 3’-gene-proximal regions and emphasize conserved PLMs in both species, particularly in the 3’-gene-proximal region. Comparison with resources from transcription factor and microRNA binding sites shows that 79% of the identified PLMs are unassigned, although some are supported by MNase-defined cistrome occupancy analysis. Enrichment analyses further reveal that unassigned PLMs provide functional predictions that differ from those derived from transcription factor and microRNA binding sites. Our study provides a comprehensive map of PLMs and demonstrates their potential utility for future characterization of orphan genes in plants.Identification of  These 3ent (CE) .A. thaliana and Z. mays are distant, the TFBSs of orthologous TFs are found to be similar in sequence  were susceptible to bind to 5’ (3’)-tPLMs in ectively Figure 4Brassica that miRNA methylates the promoter region of SP11 gene to silence it  can target transcripts with sequence complementarity , thus inlence it . Hence, lence it ”. we fouectively . We founA. thaliana had more frequent homologies with the 5’-seed region, whereas those from Z. mays had more frequent homologies with the compensatory 3’-end  and Basic leucine zipper factors (bZIP) represented the three major TF families identified in the 5’-gene-proximal region, while C2H2 zinc finger factors, Myb-related and HD-ZIP factors were the three major TF families identified in the 3’-gene-proximal region  corresponded to tPLMs in A. thaliana and Z. mays, respectively, were distinct from RNA polymerase II binding sites   (ctively) , suggestA. thaliana, one-third of the 5’-uPLMs were localized in group 1, while more than one-third of the 3’-uPLMs were localized in group 3. In Z. mays, the 5’-uPLMs were preferentially localized upstream and downstream of the three groups detected, showing a greater dispersion than that observed in A. thaliana. Furthermore, the 3’-uPLMs of Z. mays had a more balanced distribution among the different groups and downstream part than those of A. thaliana. Interestingly, the density of the 5’-uPLMs in both species was higher in the core promoter region corresponding to group 2 and known to be the locus of many regulatory events  to identify chromatin-accessible regions in developing maize ears led to the identification of 215 small (<30 bp) TF footprints distributed in total across 100,000 non-overlapping binding sites in the genome . Given tcis-regulatory players . Terms related to “Cell wall organisation” were also present in the five most enriched MapMan terms for the A. thaliana-uPLMs-containing gene set in both regions, although the protein classes identified were different. For example, we found that part of the genes encoding alpha-expansin are characterized by 5’-uPLMs signals localized after the TSS, while part of the genes encoding acyl omega-hydroxylases are characterized by 3’-uPLMs signals localized in group 1 and 2.Comparing 5’- and 3’-uPLMs-containing gene sets revealed specific terms associated with each of the two sets  defined only by the GO term “transposition”  revealed three modules of target genes referred to as G1, G2 and G3 with 7, 3 and 54 members, respectively  compared with that of A. thaliana , are conserved between species. Notably, we have shown that this context appears to be more conserved in the 3’-gene-proximal region than in the 5’-gene proximal region: 14% of 3’-PLMs shared between the two species compared to 7% of 5’-PLMs  and more generally in the whole region, opens interesting mechanistic perspectives on the role of TFs. First, they may act as activators or repressors of the transcriptional machinery  and tPLMs (up to 30%), also suggests that uPLMs could constitute a context that needs to be conserved. In this regard, the genomic significance of uPLMs is already supported by experimental maize ear MOA-seq data, suggesting that some of the identified uPLMs are potential TF footprints. It will be important to validate these uPLMs by functional assays to determine what proportion of them are indeed proximal  process Figure 8 process  contribuproteins  are majoproteins . This code novo PLMdetect method has demonstrated the richness of the gene-proximal regions and the interest in their further characterization. In particular, this work has highlighted the importance of the 3’-gene-proximal region as a major source of new knowledge and great interest for future studies.In summary, the implementation of the genome-wide and A. thaliana and Z. mays genes, respectively.TAIR10  and B73vA. thaliana and Z. mays, respectively. In total, 19,736 and 25,848 genes were analyzed for A. thaliana and Z. mays, respectively. For the 3’-gene-proximal region, similarly to what has been done for the 5’-gene-proximal region sequences, annotation of the TTS was ensured by filtering genes without a 3’-UTR region annotated. To standardize the PLM detection step, genes on forward strand were reverse-complemented. Extracted sequences were  and  bp with respect to the TTS for A. thaliana and Z. mays, respectively. Taking in consideration only annotated 3’-UTR, 20,573 and 25,199 genes were processed for A. thaliana and Z. mays, respectively.For the 5’-gene-proximal region, annotation of the TSS was ensured by filtering genes without a 5’-UTR region (in GFF3/GTF file). Genes on reverse strand were reverse-complemented to analyze all sequences in the same orientation. Extracted sequences corresponded to the intervals  and  bp relative to the TSS for Every non-polymorphic DNA 4-mers to 8-mers was generated representing 87,296 motifs. Among these motifs, 256, 1,024, 4,096, 16,384, and 65,536 had a length of 4, 5, 6, 7 and 8 bp, respectively.To check PLM redundancy, we calculated the Jaccard index of each pair of PLMs for each PLM containing-gene set with an inclusion link. This index was obtained by dividing the intersection of the two lists by their union. A Jaccard index of 0.9 indicates an almost perfect match between the gene lists. This index was also calculated on the functional window of each pair of PLMs to quantify their overlap. We set the threshold for the functional window Jaccard index at 0.5.A. thaliana  and Z. mays  were kept.TFBSs  were extracted from JASPAR Plant 2020  and ChIPWe first assigned TFBSs to PLMs for both species in each region separately using the TOMTOM web tool . EuclideP-values were adjusted by the Benjamini-Hochberg (BH) procedure to control the False Discovery Rate (FDR). An enriched term had its adjusted P-value lower than 0.05.Functional annotation of genes from both species was based on MapMan X4  and GeneZ. mays 5’-PLMs were compared to published MOA-seq motifs  and Z. mays  using a hypergeometric test with the R phyper function. P-values were adjusted by the BH procedure to control the FDR. An enriched term had its adjusted P-value lower than 0.05.Functional enrichment of each TF module was performed by comparing the relative occurrence of each term to its relative occurrence in the list of genes encoding TFs in https://forgemia.inra.fr/GNet/plmdetect/plmdetect_tool.The original contributions presented in the study are included in the article/VB, M-LM and SC conceived the project. M-LM and SC designed and supervised the study. JR generated, analyzed and interpreted the data. CG formatted the functional annotation files. JR, M-LM and SC wrote the manuscript. All authors contributed to the article and approved the submitted version.® Carnot Institute in the frame of the PLMViewer program. Plant2Pro® is supported by ANR (agreement #19 CARN 0024 01). The IPS2 and IJPB laboratories benefit from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007).This work was supported by the Plant2ProWe thank all members of the ‘Genomic Networks’ (GNet) and ‘Biomass Quality and Interactions with Drought’ (QUALIBIOSEC) teams past and present. We also thank Hank W. Bass for sharing some works prior to publication, Hervé Vaucheret for very helpful discussions, and two reviewers for comments that improved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The impact of severe PPM in patients undergoing TAV-in-TAVR is unknown.A severe prosthesis-patient mismatch (PPM) is associated with adverse outcomes following transcatheter aortic valve replacement (TAVR) for We sought to investigate the incidence and 1-year outcomes of different grades of PPM in patients undergoing TAV-in-TAVR.The TRANSIT-PPM is an international registry, including cases of degenerated TAVR treated with a second TAVR. PPM severity, as well as in-hospital, 30-day, and 1-year outcomes were defined according to the Valve Academic Research Consortium-3 (VARC-3) criteria.p = 0.04). Specifically, the rate of severe PPM was significantly higher among cases of a SE TAVR implanted into a balloon-expandable (BE) device . At 1-year follow-up, the rate of all-cause mortality, and the rate of patients in the New York Heart Association (NYHA) class III/IV were significantly higher in the cohort of patients with severe PPM . Almost all the patients with a severe PPM after the first TAVR had a failed < 23 mm BE transcatheter heart valve (THV): the treatment with an S-SE resolved the severe PPM in the majority of the cases.Among 28 centers, 155 patients were included. Severe PPM was found in 6.5% of patients, whereas moderate PPM was found in 14.2% of patients. The rate of severe PPM was higher in patients who underwent TAV-in-TAVR with a second supra-annular self-expanding (S-SE) TAVR (10%, After TAV-in-TAVR, in a fifth of the cases, a moderate or severe PPM occurred. A severe PPM is associated with an increased 1-year all-cause mortality.https://clinicaltrials.gov], identifier [NCT04500964].[ Prosthesis-patient mismatch (PPM) may occur after surgical aortic valve replacement (SAVR) or transcatheter aortic valve replacement (TAVR) when a normally functioning prosthetic valve presents an effective orifice area (EOA) relatively small for the patient’s body surface area (BSA), thus not allowing an adequate cardiac output . SeveralClinicalTrials.gov Identifier: NCT04500964). We evaluated cases performed with supra-annular self-expanding (S-SE)  and intra-annular balloon-expandable (BE) transcatheter heart valves (THVs) .The TRANSIT-PPM project is an investigator-initiated international multicenter registry, including consecutive patients undergoing TAVR for a degenerated transcatheter aortic valve  < 30 kg/m2, or > 0.55 and < 0.70 cm2/m2 for patients with BMI > 30 kg/m2, and severe if the predicted EOA was ≤ 0.65 cm2/m2 for patients with BMI < 30 kg/m2 and ≤ 0.55 for patients with BMI > 30 kg/m2  definitions . In part30 kg/m2 –10.The left ventricular outflow tract (LVOT) measures have been obtained with the CT scan that all the patients performed before the procedure.P-value of < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS software version 23 .Descriptive statistics are reported as mean and SD for normally distributed continuous variables, as median and 25–75th percentile otherwise. Absolute and relative frequencies are reported for categorical variables. For continuous variables, the comparisons were done either with ANOVA or with a non-parametric test . For categorical variables, comparisons among groups were done with the chi-squared tests or Fisher’s exact tests. All-cause death was reported using the Kaplan–Meier estimates together with their 95% CI. The Wilcoxon signed rank sum test was used for the comparison of echo parameters in paired analyzes. The cumulative incidences of clinical events at follow-up were assessed with the Kaplan–Meier method and log-rank test. A two-sided Because of the sensitive nature of the data collected for this study, requests to access the dataset from qualified researchers trained in human subject confidentiality protocols may be sent to the corresponding author.ClinicalTrials.gov Identifier: NCT04500964). A group of 28 centers took part in the project: 22 in Europe, 4 in North America, 1 in South America, and 1 in the Middle East. Among a total number of about 40,000 procedures performed since 2008, 155 cases of TAV-in-TAVR were eventually included in the TRANSIT-PPM study. Of these, 73 (47%) cases presented a degenerated supra-annular self-expanding valve, while 82 (53%) cases had a degenerated balloon-expandable device.The TRANSIT project is an investigator-initiated registry that started collecting data in January 2020  and 32 (20.6%) patients, respectively, presented a severe or moderate PPM after the first procedure, while no patients had a mean residual gradient higher than 20 mm Hg or a more than mild aortic regurgitation (AR).The mean age was 77.9 ± 7.7 years and the male gender was slightly more represented (57.4%). The majority of patients (74%) were in the NYHA class III or IV at admission. The mean left ventricular ejection fraction was 49 ± 13.4. The European System for Cardiac Operative Risk Evaluation I (EuroSCORE I) was 20.3 ± 15.0, the EuroSCORE II was 8.7 ± 7.5, and the STS score was 6.3 ± 5 . Most paPatients were grouped and analyzed according to the grade of PPM after the second TAVR: 10 (6.5%) patients had severe PPM, 22 (14.2%) patients had moderate PPM, and 123 (79.3%) patients had no PPM.p = 0.05 and p = 0.04, respectively). No other differences were found among common risk factors such as diabetes, chronic obstructive pulmonary disease (COPD), and severe renal failure .There were no differences in BSA and BMI distribution between the groups . Overall failure . Risk scp = 0.001).Of note, 4 out of 10 patients presenting a severe PPM after TAV-in-TAVR belong to the mixed-degenerated cohort (All the cases of severe PPM after the first TAVR concerned patients with a BE THV (8 patients), with a significantly higher prevalence of ≤ 23 mm THVs (7 out of 8); conversely, no grade of PPM was more frequent among patients with an S-SE THV, in particular in patients with a > 23 mm THV , 2.In this cohort of patients with a degenerated first THV undergoing TAV-in-TAVR, an S-SE THV was implanted in 86 cases (55%), while a BE THV was implanted in the remaining 69 cases (45%) (see We could not find a specific strategy in the selection of the second TAVR except at the operator’s discretion.p = 0.04) (p = 0.0001).The cohort of patients treated by means of an S-SE showed a significantly higher rate of severe PPM compared to those who received a BE  . On the p = 0.003). The rate of moderate PPM was significantly higher when a BE THV has been used to treat a degenerated BE THV   .Overall, the rates of severe and moderate PPM were significantly higher in patients presenting with a degenerated ≤ 23 mm THV : in partAll the patients with a severe PPM after the first TAVR have been treated with an S-SE: only 1 out of 8 patients had a severe PPM after the second TAVR .The VARC-3 defined procedural success rate was 80.6% with 22 (14.2%) patients presenting: a severe PPM (9 patients) and/or residual gradient ≥ 20 mm Hg (13 patients), and 2 (1.3%) patients showing a more than mild AR.p = 0.004], and of a degenerated THV of ≤ 23 mm  were independent predictors of severe PPM after the second TAVR.The presence of a no PPM after the first TAVR [hazard ratio (HR) 0.126, 0.31–0.51, p = 0.006). Two patients had a myocardial infarction during the hospital stay. No differences in the incidence of conduction disturbances, pacemaker (PM) implantation, or new-onset atrial fibrillation were observed according to the presence and severity of PPM. Other in-hospital outcomes are shown in Seven patients (4.5%) died during the in-hospital stay, all due to cardiovascular (CV) causes. None presented a severe PPM, while 4 patients had moderate PPM and 3 patients had no significant PPM (p = 0.08). Compared to patients with moderate or no PPM, those patients with a severe PPM showed a higher rate of valve-related hospitalization (p = 0.001) and dyspnea at rest or on mild exertion (the NYHA class III/IV) (p = 0.001) (p = 0.001).A 30-day cumulative overall mortality rate was 7.1% with no further cardiovascular death and no significant differences reported among groups (= 0.001) . Two casp = 0.016).Cumulative 1-year all-cause mortality was 12.9%  with a CV-related death occurring in 5.8% of patients. Compared to patients with no and moderate PPM, the rate of all-cause mortality was significantly higher in patients with a severe mismatch (p = 0.002)  .p = 0.0001). No cases of valve thrombosis, myocardial infarction (MI), stroke, or valve dysfunction requiring intervention were further recorded.Valve-related hospitalization occurred in 10 (6.5%) patients, with no significant differences between the groups. The rate of patients in the NYHA class III/IV was significantly higher in the severe PPM cohort .No differences in the rate of all-cause mortality, CV mortality, and valve-related hospitalization were found among those patients with elevated postprocedural mean gradient (≥ 20 mm Hg), but without severe PPM , particularly in those with an S-SE THV implanted into a degenerated BE THV (p = 0.003).The rate of severe PPM after TAV-in-TAVR was significantly higher in patients treated with an S-SE THV , particularly in those with a BE THV implanted into a degenerated BE THV.The rate of moderate PPM after TAV-in-TAVR was significantly higher in patients treated with a BE THV  and a degenerated ≤ 23 mm THV  are independent predictors of severe PPM after TAV-in-TAVR.A no PPM after the first TAVR  with no impact of PPM on cardiovascular mortality or the NYHA class at 1 year. Recently, an analysis of the TVT Registry, including patients undergoing TAVR with self-expanding THVs, showed a rate of severe PPM of 5.3% in patients undergoing de novo TAVR and 27% in those patients undergoing TAVR-ViV : a possible explanation for this finding might be the fact that almost all the patients presenting with a severe PPM after the second TAVR actually had a degenerated THV ≤ 23 mm. In other words, in the presence of quite small anatomy, even the supra-annular position, which is associated with a larger EOA, might not be enough to resolve the PPM . Whether this can be completely ascribed to the presence of severe PPM or is influenced by increased frailty, presence of significant comorbidity and reduced functional status as reflected by the presence of significantly higher risk scores (Finally, consistently with the available literature , 12, 13,k scores  should bThe techniques of bioprosthetic valve fracture/remodeling and BASILICA have been successfully applied to the field of ViV to reduce the risk of residual high gradient and coronary obstruction/sinus sequestration in patients with a degenerated surgical bioprosthesis , 15. TheClearly, very fragile or old patients will unlikely experience a structural valve deterioration considering their inherent risk of mortality : in thesOur data also pointed out the importance of the anatomy and, as a consequence, of the choice of the first THV, at the beginning of the “valve journey”: small anatomy is obviously the real challenge for the reintervention, as it poses a high risk of coronary obstruction/flow impairment, as well as of severe PPM.An S-SE might be associated with better durability , thus suOverall, a tailored approach at the time of the first TAVR is becoming critically important and the implementation of implantation techniques aiming at the commissure-to-commissure alignment should be pursued in every case in order to minimize the subsequent risk of coronary flow impairment and difficult coronary reaccess. Similarly, the evaluation of the risk of significant PPM, which is more likely with BE THVs, should be evaluated with the risk of PVL that, on the contrary, seems to favor the BE THVs, likewise the risk of pacemaker implantation –19.Being an investigator-initiated registry, no central adjudication of events has been performed and echo data have been collected by the participating centers. The relatively low sample size does not allow definite conclusions, indeed the latter should be viewed as hypothesis-generating; however, this is the largest series in the field of TAV-in-TAVR and the present analyzes of the PPM may serve to generate and design future studies.The rate of moderate and severe PPM after TAV-in-TAVR is lower than that observed after TAVR-ViV, but, as expected, higher than TAVR in native aortic annuli. A severe PPM is associated with increased 1-year mortality and reduced functional capacity. At the time of the first treatment, a modern approach to TAVR should consider the possible future need for a reintervention and its implications, especially when evaluating patients with long-life expectancy in whom a structural valve deterioration is likely to occur.- Following the degeneration of a THV, the procedure of TAV-in-TAVR will surely be progressively more frequent.- After a TAV-in-TAVR, the risk of severe PPM is more frequent with specific first-second THVs combinations and it is significantly more frequent when a severe PPM was present yet after the first TAVR.- A severe PPM implies a higher rate of both the 1-year mortality and the NYHA class III/IV, thus a careful evaluation should be made at the time of the first procedure, at the beginning of the “valve journey.”The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by the ECs of every participating center have been informed. The patients/participants provided their written informed consent to participate in this study.MC and EC drafted the manuscript. LT and FB provided expert revision. MC performed the statistical analysis. All authors contributed to the collection of the data and provided critical comments to the manuscript.


Tobacco use is the leading cause of preventable death worldwide, and relapse during abstinence remains the critical barrier to successful treatment of tobacco addiction. During abstinence, environmental contexts associated with nicotine use can induce craving and contribute to relapse. The insular cortex (IC) is thought to be a critical substrate of nicotine addiction and relapse. However, its specific role in context-induced relapse of nicotine-seeking is not fully known. In this study, we report a novel rodent model of context-induced relapse to nicotine-seeking after punishment-imposed abstinence, which models self-imposed abstinence through increasing negative consequences of excessive drug use. Using the neuronal activity marker Fos we find that the anterior (aIC), but not the middle or posterior IC, shows increased activity during context-induced relapse. Combining Fos with retrograde labeling of aIC inputs, we show projections to aIC from contralateral aIC and basolateral amygdala exhibit increased activity during context-induced relapse. Next, we used fiber photometry in aIC and observed phasic increases in aIC activity around nicotine-seeking responses during self-administration, punishment, and the context-induced relapse tests. Next, we used chemogenetic inhibition in both male and female rats to determine whether activity in aIC is necessary for context-induced relapse. We found that chemogenetic inhibition of aIC decreased context-induced nicotine-seeking after either punishment- or extinction-imposed abstinence. These findings highlight the critical role nicotine-associated contexts play in promoting relapse, and they show that aIC activity is critical for this context-induced relapse following both punishment and extinction-imposed abstinence. Tobacco use is one of the leading causes of preventable death worldwide. In both abstinent and nonabstinent individuals with a history of nicotine use, exposure to cues associated with nicotine use provokes craving , which iThe insular cortex (IC) has been considered a critical neural substrate of nicotine addiction since it was discovered that some human patients with stroke-induced damage to their insula had a higher probability of smoking cessation . SubsequHere, we demonstrate for the first time, in both male and female rats, context-induced relapse of nicotine-seeking after punishment of nicotine taking in an alternative context. Using the neuronal marker of activity Fos , we showIn this experiment, we aimed to identify total activity in IC and BLA, and the inputs to aIC, that are associated with context-induced relapse after punishment. F = 16.1, p < 0.001), indicating that responses on the active nose-poke increased throughout training compared to inactive nose-pokes. In punishment  = 9.6; p < 0.001), reflecting the decrease in active nose-pokes during punishment. On the final test, we returned the rats to either context B (Punishment) or context A (Nicotine)  = 14.7; p < 0.01), and a Test Context × Nose-Poke interaction  = 6.4; p < 0.05). These data show that rats tested in the nicotine context significantly increased nicotine-seeking (active nose-pokes) compared to the rats tested in the punishment context.Statistical analysis of the training data  revealednishment , we obseicotine) . We founipsilateral to CTb injection. Using separate one-way analysis of variance (ANOVA) for each region, we found a significant effect of Test Context for Total Fos in aIC  = 13.1; p = 0.001), mIC  = 12.6; p = 0.001), and pIC  = 10.1; p = 0.002). Subsequent Tukey post hoc only revealed a significant difference between rats tested in nicotine versus punishment context for aIC (p = 0.025). We found no effect of Test Context for the total CTb in mIC and pIC . For the percent of CTb neurons that also express Fos, there was a main effect of Test Context in both mIC  = 5.3; p < 0.05) and pIC  = 6.5; p < 0.05). However, post hoc analysis revealed no significant difference between the Nicotine and Punishment tested rats.contralateral to CTb injection. Using separate one-way ANOVA for each region, we found a comparable pattern of effects for Total Fos. Specifically, there was a main effect of Test Context in aIC  = 14.1; p < 0.001), mIC  = 8.3; p < 0.01), and pIC  = 21.6; p = 0.002), and subsequent post hoc revealed a significant difference between rats tested in nicotine or punishment context in aIC (p = 0.02) and pIC (p = 0.003), but not mIC (p = 0.32). In all three regions we found no effect of Test Context on total CTb . For the percent of CTb neurons that also express Fos, there was a main effect of Test Context in both aIC  = 7.4; p < 0.01) and mIC  = 4.2; p < 0.05), but not pIC  = 1.8; p > 0.05). Post hoc analysis revealed significant difference between the Nicotine and Punishment tested rats in aIC (p = 0.01) but not mIC (p = 0.3). These data show that context-induced relapse of nicotine-seeking is associated with increased activity in the aIC neurons that project to the contralateral aIC.ipsilateral to the CTb injection. No CTb was observed in the contralateral BLA. Anterior and posterior BLA  have distinct connectivity and functions  = 21.1; p < 0.001) and posterior BLA  = 9.4; p < 0.001). Subsequent Tukey post hoc revealed a significant difference between rats tested in nicotine or punishment context in aBLA (p < 0.001) and pBLA (p = 0.007). We found no effect of Test Context on total CTb in both aBLA  < 1; p > 0.05) and pBLA  < 1; p > 0.05). Finally, analysis of the percent of CTb-positive neurons that are also Fos positive revealed a significant effect of Test Context in aBLA  = 13.5; p < 0.001) and pBLA  = 4.6; p < 0.05). Tukey post hoc analysis revealed a significant difference between rats tested in nicotine versus punishment context in aBLA (p = 0.001) but not pBLA (p = 0.1). In summary, these data show that context-induced relapse of nicotine-seeking is associated with increased activity in BLA, and that there is also selectively increased activity in the aBLA → aIC pathway.In unctions , and wer2+) transients throughout the entire task. 2+ sensor jGCaMP7f  = 12.8, p < 0.05). In punishment  = 1.0, p > 0.05). We returned the rats to context A (Nicotine) on the final two test sessions in consecutive days  = 15.8; p < 0.01) reflecting greater responses on the active nose-poke compared to the inactive nose-poke in these test sessions.Repeated measures ANOVA on the self-administration data  revealednishment  repeatedive days . We foun2+ transients after nose-pokes. Bootstrapping analysis revealed a significant increase from baseline for nicotine-reinforced active nose-pokes (−0.04 to +8.56 s), nonreinforced active nose-pokes (+0.1 to +5.1 s), and inactive nose-pokes (+0.3 to +4.1 s). The permutation tests comparing the activity of each of these outcomes also revealed significant differences between them (p < 0.01). The most relevant observation is that calcium activity after a reinforced active nose-poke was significantly higher than a nonreinforced active nose-poke . This reflected a biphasic excitatory Ca2+ response indicating increased activity in aIC related to the nicotine-associated cue and nicotine infusion. Another potentially interesting observation is that there was a significant difference between nicotine-reinforced active and inactive nose-pokes prior to the response (−0.67 s to 0) suggesting a role for aIC in encoding response–outcome contingencies.In self-administration, we found that aIC has significant excitatory Caf/f to represent the change in signal from before the nose-poke to after. These data show a significant increase in aIC activity after each response type throughout self-administration.In punishment we again found that aIC shows excitatory transients after all nose-pokes. Bootstrapping analysis revealed a significant increase from baseline for unpunished nicotine-reinforced active nose-pokes (+0.08 to +3.28 s) and for nonreinforced active nose-poke (−0.54 to +6.86 s), although comparisons did not indicate a difference between them. We also observed a significant increase after inactive nose-poke (+0.15 s to +1.78). The change in calcium activity to punished active nose-pokes was much more pronounced, lasting the entire epoch of analysis (−0.17 s to +10 s).Permutation tests comparing the activity of each of these outcomes also revealed some interesting patterns of activity (p < 0.008). First, there was no significant difference between unpunished nicotine-reinforced active nose-pokes and nonreinforced active nose-pokes. This reflects a change from the nicotine self-administration phase, as there was no selective encoding of nicotine infusion compared to nonreinforced active nose-pokes. Second, punished active nose-pokes were significantly higher than both unpunished nicotine-reinforced and nonreinforced active nose-pokes (+0.33 to +10 s), reflecting the profound response of aIC to footshock punishment. As observed during nicotine self-administration training, calcium activity in aIC was significantly higher prior to the nose poke for active responses  compared to inactive nose-pokes (−0.73 to 0 s), indicating that aIC activity selectively increases for active nose-pokes prior to the response.f/f to represent the change in signal from before the nose-poke to after. These data show a significant increase in aIC activity after each response type throughout punishment.In the final two sessions we tested the rats in the original training context (context A) under extinction conditions over 2 consecutive days. Bootstrapping analysis revealed a significant increase from baseline for active nose-pokes that led to the nicotine-associated cue (−0.54 to +3.41 s), nothing (−0.17 to +4.35 s), and the inactive nose-pokes (+0.21 to +3.97 s). This surprisingly consistent pattern of activity for all types of nose-pokes was reflected in the permutation tests (p < 0.01), which revealed no significant difference between the three types of outcomes following the nose-poke. However, like in the previous phases, permutation tests revealed a significant difference between active nose-pokes (leading to nicotine-associated cue) and inactive nose-pokes prior to the response (−0.61 s to 0).f/f to represent the change in signal from before the nose-poke to after. These data show a significant increase in aIC activity after each response type in these test sessions.We first determined the validity of our chemogenetic inhibition approach and used ex vivo slice physiology in aIC . RecordiGiven the observations of increased Fos in aIC associated with context-induced relapse, and task-related activity in aIC, we aimed to determine whether activity in aIC is necessary for context-induced relapse after punishment-imposed abstinence. F = 13.4, p < 0.001), with responses on the active nose-poke increasing throughout training compared to inactive nose-pokes. F = 7.1, p < 0.001), with responses on the active nose-poke decreasing throughout punishment compared to inactive nose-pokes. F = 13.2; p = 0.001), and a Context × Group × Nose-Poke interaction  = 11.6; p = 0.001). We observed no overall effect of Sex  = 4.0; p > 0.05). Moreover, we found no Sex × Group × Nose-Poke interaction  = 2.5; p > 0.05), nor Sex × Context × Group × Nose-Poke interaction  = 3.6; p > 0.05). These results show that chemogenetic inhibition of aIC significantly decreased context-induced relapse of nicotine-seeking after punishment, in both male and female rats.F < 1; p > 0.05). Next we analyzed the data from the latency to the first nose-poke on the inactive nose-poke during the test sessions  = 1.4; p > 0.05), nor Context × Group interaction  < 1; p > 0.05). While indirect, each of these measures indicates that the observed reduction in active nose-pokes does not occur alongside a broader, nonspecific reduction in behavioral output.We conducted two follow-up statistical tests specifically on the inactive nose-pokes to further test for potential nonspecific reductions in behavior caused by the off target effects of CLZ. First we ran a post hoc repeated measures ANOVA on the inactive nose-pokes, and found no Context × Group interaction  = 12.3, p < 0.001), indicating that responses on the active nose-poke increased throughout training compared to inactive nose-pokes. F = 14.0; p < 0.001), and Nose-Poke  = 15.6; p < 0.001), but no Session × Nose-Poke interaction  = 1.5; p > 0.05); both the active and inactive nose-poke decreased throughout extinction. F = 8.5; p < 0.01), as well as a Context × Group × Nose-Poke interaction  = 11.3; p < 0.01). We observed no overall effect of Sex  = 1.6; p > 0.05), and no Sex × Group × Nose-Poke interaction  = 1.1; p > 0.05), nor Sex × Context × Group × Nose-Poke interaction  < 1; p > 0.05). These results show that chemogenetic inhibition of aIC significantly decreases context-induced relapse of nicotine-seeking after extinction, in both male and female rats.F = 1.5; p > 0.05). Next we analyzed the data from the latency to the first nose-poke on the inactive nose-poke during the test sessions  < 1; p > 0.05), nor Context × Group interaction  < 1; p > 0.05). While indirect, each of these measures indicates that the observed reduction in active nose-pokes does not occur alongside a broader, nonspecific reduction in behavioral output.We again conducted two follow-up statistical tests specifically on the inactive nose-pokes to further test for potential nonspecific reductions in behavior caused by the off target effects of CLZ. First we ran a post hoc repeated measures ANOVA on the inactive nose-pokes, and found no Context × Group interaction  because CNO is converted to CLZ in vivo  and chemogenetic inhibition (hM4Di: mCaMKIIa). Therefore, the observations reported in the photometry experiment likely include activity from a population of neurons that were not manipulated in the chemogenetics experiments. It will be of interest in future studies to identify potential variation in the responses of aIC neuronal subpopulations to nicotine infusions and punishment. Finally, in this experiment we cannot determine whether the change in aIC activity in response to the various outcomes in this experiment were dependent on a preceding action , or whether noncontingent outcomes would yield the same result. It will be of interest to measure aIC activity during noncontingent shock delivery, nicotine infusions, and nicotine-associated cues.In preclinical studies, the role of aIC in drug-seeking and relapse is well established . BilaterOur results also show increased activity in BLA during context-induced relapse after punishment-imposed abstinence. The role of BLA in cue- and stress-induced reinstatement of nicotine-seeking has been demonstrated previously , but to We also found increased Fos in contralateral aIC projections during context-induced relapse. Corticocortical pathways are primarily thought to result in feedforward inhibition through targeting of the PV interneurons in the contralateral hemisphere . The conOur results using fiber photometry revealed increased aIC neural activity after nose-pokes in all phases of the task. While this suggests that aIC activity may be generally important in encoding response–outcome contingencies, we observed important differences in the patterns of activity in the different phases. In self-administration, aIC activity was highest following nose-pokes that lead to nicotine infusions. During punishment, the response to nonreinforced active nose-pokes changed such that there was no longer a difference between nicotine reinforced nose-pokes and nonreinforced active nose-pokes. This may reflect increased uncertainty of the outcome, as active nose-pokes during this phase could lead to nicotine infusion or nicotine infusion plus shock. Alternatively, it may reflect a change in the motivational determinant of behavior from appetitive to aversive. Indeed, aIC activity in response to the punished nose-pokes was significantly higher than all other outcomes. Such a change in the response of aIC to (nonpunished) nicotine infusion in punishment may reflect a reevaluation of nicotine reward encoding within aIC because the punishment overcomes the motivation for nicotine and suppresses nicotine-seeking. The observed reduction in nicotine-seeking in punishment is an adaptive response, thus we propose that during operant behavior aIC activity may be involved in the adaptation of behavior in response to both rewarding  and aversive outcomes . It will be of interest in future studies to determine whether maladaptive responses to punishment, such as the punishment-resistance phenotype , are assThe IC is known to support a general function of integrating interoceptive information , which cWe also found that aIC activity increased relative to baseline prior to an active nose-poke, but not inactive nose-pokes. This pattern of activity was consistent throughout the experiment. Other studies have shown that activity in aIC is necessary for the performance of goal-directed behavior , but it The observation that aIC activity in the relapse tests did not specifically relate to the response type or outcome. While this may be potentially inconsistent with the finding that chemogenetic inhibition decreases context-induced relapse, we think that the use of contextual cues to induce reinstatement may explain this. Photometry analyses are better suited to detecting phasic, rather than background changes in activity, which may be why we did not identify a specific signal related to relapse in the aIC photometry data. Fos studies are better suited for this, and we did observe increased Fos in aIC during context-induced relapse. Our aim in this study was to keep the behavioral task consistent across experiments; it will be of interest in future studies to test whether cue-induced reinstatement , or triaDistinct learning mechanisms are responsible for behavioral control after extinction or punishment. Both are mediated by new context-dependent associations . HoweverWe sought to investigate the neural substrates of context-induced relapse to nicotine-seeking after punishment-imposed abstinence. Our results show that activity in aIC is necessary for context-induced relapse of punished nicotine-seeking, and this is also the case for extinguished nicotine-seeking. We also show that the BLA projections to aIC are activated during context-induced relapse, and future studies are needed to determine the extent to which this activity is necessary for this relapse. Our findings further highlight the critical importance of the anterior IC as a target for nicotine addiction treatments.We obtained 91 Wistar rats , aged 10–12 weeks upon arrival, from Charles River Laboratories B.V. . In compliance with Dutch law and Institutional regulations, all animal procedures were approved by the Centrale Commissie Dierproeven  and conducted in accordance with the Experiments on Animal Act. Experiments were approved by the local animal welfare body Animal Experiments Committee of the Vrije Universiteit, Amsterdam, The Netherlands. Behavioral tests were conducted during the dark phase of the rat’s diurnal cycle (12 hr/12 hr). Food and water were available ad libitum, and rats were single housed the rats after surgery for the remainder of the experiment.We did not make a specific power analysis to determine sample size prior to any experiments. The group size was chosen based on our past research , suggestAll procedures were performed in standard Med Associates operant chambers with data collected through the MED-PC IV program . Each chamber had one ‘active’ and one ‘inactive’ nose-poke hole on one wall and a grid floor connected to shock controllers. Contexts A and B were defined by houselight (on/off), cue-light color (white/red), and white noise (on/off).The catheter for intravenous nicotine delivery was composed of a cannula connector pedestal , attached to a 95-mm silicone catheter  and a 6-mm piece of polyethylene tubing  clamping the silicone catheter to the connector pedestal. A small ball of silicone (RTV-1 Silicone Rubber/Elastosil ) is attached 38 mm from the end of the silicone catheter.https://github.com/ialozares/NicInsulaPhotometry, copy archived at swh:1:rev:6d3efefa0f7e379c2f5bdccc1f3ca70d225bce5e . Blue excitation light  was modulated at 211 Hz and passed through a 460–490 nm filter (Doric Lenses), while isosbestic light  was modulated at 531 Hz and passed through a filter cube (Doric Lenses). GCaMP7f fluorescence was passed through a 500- to 550-nm emission filter (Doric Lenses) and onto a photoreceiver (Newport 2151). Light intensity at the tip of the fiber was measured before every training session and kept at 21 µW. A real-time processor  controlled excitation lights, demodulated fluorescence signals and received timestamps of behavioral events. Data were saved at 1017.25 Hz and analyzed with custom-made Matlab scripts, available at 225bce5e .Nicotine  was dissolved in normal saline, filtered, and pH adjusted to 7.4. CLZ was dissolved first in a small amount of glacial acetic acid  and progressively diluted in saline until a final concentration of 0.3 mg/ml (pH was adjusted to 7.0–7.2).hM4Di), AAV-5/2-mCaMKIIa-EGFP-WPRE-hGHp(A) (GFP), and AAV-9/2-hSyn1-chI-jGCaMP7f-WPRE-SV40p(A) (jGCaMP7f). The titer injected was: hM4Di, 2.4 × 1012 gc/ml; GFP, 2.5 × 1012 gc/ml; jGCaMP7f, 4.4 × 1012 gc/ml.We purchased premade viral vectors from the University of Zurich viral vector core: AAV-5/2-mCaMKIIa-HA_hM4D(Gi)-IRES-mCitrine-WPRE-hGHp(A)  and the antibiotic Baytril . Surgery was performed under isoflurane gas anesthesia . The silicone catheter was tunneled from the scalp to the neck and was inserted into the jugular vein, where it was secured using sterile thread. We sealed the silicone catheter using a taurolidine-citrate solution  and a polyethylene cap. After the catheter was implanted, we placed the rat in a stereotactic frame  and injected xylocaine 2% with adrenaline  into the incision site prior to the incision. A craniotomy above aIC was performed, followed by CTb or AAV injections (see below for details). After filling the skull hole with bone wax, cannula tubing connected to a Plastics One Connector-Pedestal and optic fiber implant (when applicable) was secured to the skull using dental cement  and jewelers screws. Rymadil  was administered for 2 days after the surgery. Rats were given 1 week of recovery following surgery.40 nl of 1% CTb  was injected unilaterally (left or right) into aIC  over 2 min using 1.0 µl 32 gauge ‘Neuros’ syringe (Hamilton) attached to a UltraMicroPump (UMP3) with SYS-Micro4 Controller (World Precision Instruments). The needle was left in place for an additional 2 min after injections.0.5 µl of AAV solution was injected unilaterally (left or right) into aIC  over 5 min. The needle was left in place for an additional 5 min. A 400-μm optic fiber (Doric Lenses) was then implanted above aIC .1.0 µl of AAV solution was injected bilaterally into aIC  over 5 min using 10 µl Nanofil syringes (World Precision Instruments), with 33-gauge needles, attached to a UltraMicroPump (UMP3) with SYS-Micro4 Controller (World Precision Instruments). The needle was left in place for an additional 5 min.On the day prior to self-administration, before and after each self-administration session and during weekends, rat’s catheters were flushed with approx. 0.1 ml mixture of heparin  and gentamicin sulfate . Rats were trained to self-administer nicotine in 2-hr sessions, 5 days a week. Entry into the active nose-poke resulted in intravenous nicotine delivery infused over approximately 2 s (infusion time adjusted for weight) at 30 µg/kg/infusion. Nicotine infusion was paired with a 20-s time-out period with the cue-light on. During time-out, responses were recorded but had no consequence. Inactive nose-pokes had no effect in either context. Rats were first trained on a fixed-ratio (FR) 1 schedule, which was then increased to FR2, followed by FR3. We tested catheter patency using intravenous anesthetic 0.05 cc pentothal .Nicotine self-administration was maintained on the FR3 schedule, and 50% of the reinforced active nose-pokes (pseudo-randomly determined by the Med-PC program) resulted in footshock (0.30 mA for 0.5 s) and nicotine infusion.Entry into the active nose-poke resulted in simultaneous activation of the cue-light and delivery of the same volume of saline through the jugular catheter (FR3 schedule).Following abstinence (PUN or EXT), rats were tested in contexts A and B. The response-contingent CS was presented on an FR3 schedule without punishment, saline or nicotine delivery. For the CTb +Fos experiment (Exp. 1), rats were tested for one 60-min session and perfused 90 min after the beginning of the test. For the chemogenetics experiments (Exp. 3 and 4) rats were tested in both contexts A and B . 30 min prior to the relapse test sessions, we injected both GFP and hM4Di expressing rats with CLZ at a dose of 0.3 mg/kg injection (i.p.). To habituate the rats to an i.p. injection, we injected approx. 0.1 ml saline i.p. prior to two nicotine self-administration sessions in context A, and two abstinence sessions (punish or extinction) in context B.We deeply anesthetized rats with isoflurane and Euthasol injection (i.p.) and transcardially perfused them with ~100 ml of normal saline followed by ~400 ml of 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.4). The brains were removed and postfixed for 2 hr, and then 30% sucrose in 0.1 M phosphate-buffered saline (PBS) for 48 hr at 4°C. Brains were then frozen on dry ice, and coronal sections were cut (40 µm) using a Leica Microsystems cryostat and stored in 0.1 M PBS containing 1% sodium azide at 4°C.Immunohistochemical procedures are based on our previously published work . We selePrimary antibodies were rabbit anti-c-Fos  and goat anti-CTb . Secondary antibodies were donkey anti-rabbit Alexa Fluor 594  and donkey anti-goat Alexa Fluor 488 .Primary antibodies were mouse anti-NeuN primary antibody  and rabbit anti-GFP primary antibody . Secondary antibodies were donkey anti-mouse DyLight 649  and donkey anti-rabbit Alexa Fluor 594 .Primary antibody was rabbit anti-GFP primary antibody  and secondary antibody was donkey anti-rabbit Alexa Fluor 594 . In rats in the GFP group, slices were only stained with DAPI.Slides were all imaged on a VectraPolaris slide scanner (VUmc imaging core) at ×10 magnification. For the CTb + Fos experiment (Exp. 1), images from Bregma +4.0 to Bregma −3.3 were scanned and imported into QuPath for analysis . For phoRegions of interest were manually labeled across sections using DAPI for identification of anatomical landmarks and boundaries. For the IC, we labeled 18 sections per hemisphere per rat spaced approximately 400 µm apart. For analysis, we separated IC into three regions anterior (aIC), middle (mIC), and posterior (pIC), and each value was the result of an average of each count from six adjacent sections: aIC (approx. Bregma + 3.72 to+ 1.44), mIC (approx. Bregma +1.08 to −0.72), pIC (approx. Bregma −1.08 to −2.92). For BLA, we labeled six sections spaced approximately 200 µm apart, and we separated it into anterior BLA (aBLA) and posterior BLA (pBLA), which is the average value of three adjacent sections: aBLA (approx. Bregma −1.92 to −2.40), pBLA (approx. Bregma −2.64 to −3.12). Some rats had missing sections due to mistakes during the process, and these sections were left blank for the statistical analyses. The contralateral BLA was not counted because we found no CTb-labeled projections from contralateral BLA into aIC.2. To identify CTb + Fos cells, each region of interest was exported to ImageJ. The overlays representing the cells (CTb or Fos) were then filled, converted to a binary layer, and then multiplied using the ImageJ function ‘Image calculator’. The nuclei that remained as a result of this function were counted as double-labeled CTb + Fos neurons. CTb + Fos double labelling is reported as a percentage of total CTb neurons for that given region of interest.To identify Fos- and CTb-positive cells, we used the ‘Cell detection’ feature in QuPath, with an identical threshold applied across all sections. CTb was not counted for the first six sections in the ipsilateral aIC, where the CTb injection was located, because the cell detection feature could not reliably discriminate between CTb-positive cell and the CTb injection. The total number of positive cells per region was divided by the area in mmN-methyl-D-glucamin (NMDG) solution containing (in mM): NMDG 93, KCl 2.5, NaH2PO4 1.2, NaHCO3 30, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 20, glucose 25, sodium ascorbate 5, sodium pyruvate 3, MgSO4⋅2H2O 10, CaCl2 ⋅ 2H2O 0.5, at pH 7.3 adjusted with 10 M HCl. The brains were removed and incubated in ice-cold NMDG solution. 300 μm thick brain slices were cut in ice-cold NMDG solution and subsequently incubated for 15–30 min at 34°C.Coronal slices were prepared for electrophysiological recordings. Rats were anesthetized  and perfused with ice-cold 2/5% CO2) ACSF solution containing (in mM): NaCl 125, KCl 3, NaH2PO4 1.2, NaHCO3 25, glucose 10, CaCl2 2, MgSO4 1. For voltage- and current-clamp experiments borosilicate glass patch-pipettes (3–5 MΩ) were used with a K-gluconate-based internal solution containing (in mM): K-gluconate 135, NaCl 4, MgATP 2, phosphocreatine 10, GTP  0.3, EGTA 0.2, HEPES 10 at a pH of 7.4. Data were sampled using a Multiclamp 700B amplifier (Axon Instruments) and pClamp software (Molecular Devices). All recordings were made between 31.1 and 33.6°C.Before the start of experiments, slices were allowed to recover for at least 1 hr at room temperature in carbogenated (95% On = 7) was tested in the nicotine self-administration context (context A), one group (n = 8) was tested in the punishment context (context B), and a third group (n = 3) was taken from the home-cage without test. We perfused rats 90 min after the start of the 60-min test. We excluded 1 rat from the experiment due to a lack of CTb expression.n = 3) or 10 days , one rat only received 4 days of punishment. In this phase 50% of nicotine infusions were paired with electric shock. After punishment-imposed abstinence, nicotine-seeking was tested in context A in 2 × 1-hr sessions over 2 consecutive days. We did not perform a context B test because we did not expect there to be enough events  to obtain a reliable aIC activity readout.Six rats were unilaterally injected with 1.0 µl of AAV encoding the inhibitory DREADD hM4Di into aIC . Rats were sacrificed 4–5 weeks later for ex vivo physiology.All behavioral data were analyzed using IBM SPSS V21. Phases were analyzed separately. Dependent variables were the total number of active and inactive nose-pokes across phases, and nicotine infusions for nicotine self-administration and punishment phases. For the CTb + Fos test (Exp. 1) we used a repeated measures ANOVA with Nose-Poke  as a within-subjects factor and Test Context (contexts A and B) as the between-subjects factor. To analyze CTb + Fos expression, we used one-way ANOVA to test for an effect of Test Context  on Fos, CTb, and % CTb + Fos/CTb. Follow-up tests (Tukey) were conducted on regions that had a significant main effect of Test Context. For the chemogenetic experiments we used repeated measures ANOVA with Test Context (contexts A and B) and Nose-Poke  as within-subjects factors, and Virus  and Sex  as between-subjects factors.F/F was then calculated as follows: ΔF/F = /fitted 405 nm signal. The resulting ΔF/F was then detrended via a 90-s moving average, and low-pass filtered at 3 Hz. ΔF/F from 5 s before nose-poke (baseline) to 10 s after nose-poke were c-ollated. These traces were then baseline corrected and converted into z-scores by subtracting the mean baseline activity during first 4 s of the baseline and dividing by the standard deviation of those 4 s. To avoid duplicate traces due to overlapping epochs, we excluded from the analyses any nose-pokes that occurred within 20 s after a rewarded nose-poke , and unrewarded active/inactive nose-pokes that occurred 5 s after another active/inactive nose-poke.Recorded signals were first downsampled by a factor of 64, giving a final sampling rate of 15.89 Hz. The 405 nm isosbestic signal was fit to the 490 nm calcium-dependent signal using a first-order polynomial regression. A normalized, motion-artifact-corrected ΔNose-poke traces were grouped by response type: active rewarded, active punished, active nonrewarded, and inactive. Two analysis approaches were used, bootstrapping and permutation tests, the rationale for each is described in detail in F/F = 0). A distribution of bootstrapped means were obtained by randomly sampling from traces with replacement . A 95% confidence interval was obtained from the 2.5th and 97.5th percentiles of the bootstrap distribution, which was then expanded by a factor of sqrt(n/(n − 1)) to account for narrowness bias  to obtain a p value per time point. Alpha of 0.05 was Bonferroni corrected based on the number of comparison conditions, resulting in alpha of 0.01 for comparisons between self-administration and test sessions (three conditions) and alpha of 0.008 for punishment sessions (four conditions). For both bootstrap and permutation tests, only periods that were continuously significant for at least 0.25 s were identified as significant .f/f presentation of the data (f/f for the 2 s prior to the nose-poke (‘−2 → 0 s’) and the 5 s after the response (‘0 → 5 s’). These values were then compared in a within-subjects t-test to determine whether there was a significant change in activity in this specific time window.For the mean dthe data  we calcu This manuscript is of interest to readers in the fields of drug addiction and relapse, reinforcement learning and punishment, and those interested in cortical functions, particularly the insular cortex. The authors extend a context and punishment-based relapse model to the widely-used drug nicotine and use a number of carefully controlled complementary approaches ranging from chemogenetic interventions to fiber photometry to support the conclusion that the insular cortex plays a role in nicotine relapse. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Michael Taffe as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Role of anterior insula cortex in context-induced relapse of nicotine-seeking" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Further analyses and precisions are needed for the photometry approach. Individual reviews provided by all referees suggest multiple lines of improvements. While we recognize that it may be difficult to address each issue, it is expected that the paper is substantially strengthened here by providing statistical analyses, possibly new angles to look at the data and by adding on the discussion of the approach and how it complements with the DREADD experiments.2) Given that no vehicle-treated groups were included in the study to formally rule out any off-site effect of clozapine, further caution may be needed to interpret the DREADD experiments. See referee 3 in particular.3) The rationale for focusing on the contralateral aIC and ipsilateral BLA inputs needs to be more clearly explained.Reviewer #2 (Recommendations for the authors):1. In experiment 1 the authors focused their analysis specifically to contralateral aIC and ipsilateral BLA inputs. Why did the authors focused their analysis on these two specific inputs? Did the authors look at over inputs? In the same experiment, the quantification of CTb and fos staining has been performed in the anterior and posterior BLA. Are there any anatomical or functional justifications for this anteroposterior segregation?2. The authors made a significant effort to characterize the coding properties of aIC neurons during nicotine self-administration, punishment, and context-induced relapse. However, several concerns are raised in the analysis of the calcium signal.For each of the 3 phases of the task  the authors report a peri-event analysis of the calcium signal between pre and post nose-poke averaging the signal across the sessions. However, it cannot be ruled out that the calcium signal change between sessions  and within sessions (i.e: comparison between first and last nose-poke of self-administration session or relapse test). This deep analysis would strengthen the results and would bring a deeper understanding of the coding properties of aIC neurons between the different phases of the task. Finally, a correlational analysis between the calcium signal and the behavioral measures  would reinforce the conclusions.3. In line with the previous comment, more details about the fibre photometry recording experiments would be helpful. Were the rats habituated to be tethered before to starting the procedure? State in the methods that the recording was performed every day of the task. Moreover, in the fibre photometry experiment the sessions of self-administration and punishment are reduced to 1h compared to the other experiments with 2-h/session. This difference suggests that the rats received less nicotine intake and context/cues exposure, it should be acknowledged. Did the authors compare the average nicotine intake between the experiments?4. Concerning the representation of the calcium signal differences. To help the reader appreciate the variation of signal I would suggest representing the most relevant comparisons as a histogram of the average of the calcium transient .5. Although discussed in the paper one important limitation in the methodology and the interpretation of the results is the lack of consistency between the promoteor of the viral vector used. In the fibre photometry experiments, the authors used hSyn promoteor suggesting that they recorded the calcium signal from all neurons of the aIC (glutamatergic neurons and GABAergic interneurons) whereas in the chemogenetic experiments they used CaMKII promoteor allowing to specifically target the glutamatergic neurons. Fiber photometry recording using CaMKII promoteor would strengthen the results and the interpretation of the coding and causal properties of the aIC neurons.6. The authors made an effort to include both male and female rats in their study. However, both sexes are not included in all the experiments making difficult to conclude about presence or lack of sex differences. It should be at least acknowledged.Reviewer #3 (Recommendations for the authors):This is an excellent paper and the authors should consider addressing the two points listed in the weaknesses section.1. The authors should consider discussing the limitations of interpretation of the fibre photometry data, particularly in regards to alternative interpretations of the increase in activity in the active nose poke -> punished condition and how this may be due to the multiple outcomes (nicotine infusion + punishment). It maybe useful to indicate on the graphs when the footshock and nicotine infusions took place.2. The authors should consider testing if there is an effect on responding with chemogenetic inhibition of the anterior insular cortex in a procedure where responding is high (e.g. nicotine or food self-administration). Interpretation of the current data in Context B could be confounded by a floor effect. Essential revisions:1) Further analyses and precisions are needed for the photometry approach. Individual reviews provided by all referees suggest multiple lines of improvements. While we recognize that it may be difficult to address each issue, it is expected that the paper is substantially strengthened here by providing statistical analyses, possibly new angles to look at the data and by adding on the discussion of the approach and how it complements with the DREADD experiments.In the revised manuscript we have provided more detailed descriptions of the statistical effects in the Results section. We have also included the raw output from the bootstrap analysis  and permutation tests  in the updated raw data files. We have also added some points in the discussion to better describe the link between the photometry data and the DREADD experiments, and also point out the strong overlap between the Fos data and these experiments.Finally, we also performed two additional analyses on the photometry data (in the supplementary data). First, we show the photometry data from the different FR stages of self-admin , the first punishment session, and the first and second relapse test. Second we calculated the mean df/f for the different events and plotted them to illustrate the individual variability between the rats in each phase.2) Given that no vehicle-treated groups were included in the study to formally rule out any off-site effect of clozapine, further caution may be needed to interpret the DREADD experiments. See referee 3 in particular.We agree that there is potential for non-specific effects of clozapine in our experiments, and have updated the discussion to address this. To further attempt to address this, we perform additional statistical analysis on the inactive nose-pokes and found no effect of clozapine, despite the rate of inactive nose-pokes being relatively high (going some way to rule out floor effects).3) The rationale for focusing on the contralateral aIC and ipsilateral BLA inputs needs to be more clearly explained.We have explained this rationale in the revised manuscript. We choose to focus on the most prominent inputs to the aIC, the more caudal regions of IC and the BLA. Only ipsilateral BLA was counted because we found no projections to aIC from contralateral BLA, which is consistent with the literature. We have revised the manuscript to more carefully explain these points.Reviewer #2 (Recommendations for the authors):1. In experiment 1 the authors focused their analysis specifically to contralateral aIC and ipsilateral BLA inputs. Why did the authors focused their analysis on these two specific inputs? Did the authors look at over inputs? In the same experiment, the quantification of CTb and fos staining has been performed in the anterior and posterior BLA. Are there any anatomical or functional justifications for this anteroposterior segregation?We have not focused on other inputs in this study. We report the anterior and posterior BLA because we counted 6 sections per rat in the AP axis (range approx. Bregma -1.92 to -3.12), and there are studies demonstrating differences in both anatomy and function in anterior versus posterior BLA. In the revised manuscript we describe some of these findings.2. The authors made a significant effort to characterize the coding properties of aIC neurons during nicotine self-administration, punishment, and context-induced relapse. However, several concerns are raised in the analysis of the calcium signal.For each of the 3 phases of the task  the authors report a peri-event analysis of the calcium signal between pre and post nose-poke averaging the signal across the sessions. However, it cannot be ruled out that the calcium signal change between sessions  and within sessions (i.e: comparison between first and last nose-poke of self-administration session or relapse test). This deep analysis would strengthen the results and would bring a deeper understanding of the coding properties of aIC neurons between the different phases of the task. Finally, a correlational analysis between the calcium signal and the behavioral measures  would reinforce the conclusions.We agree with the reviewer that there are many potential advantages to a more refined analysis. In the revised manuscript we report analysis of the different FR stages of self-admin , as well as the first punishment session, and the first and second relapse test. Due to the variability of the data, and statistical limitations, we cannot go further than this, for example looking within sessions (e.g. first vs. last nose-poke), or the last punishment session because of the low number of recorded events.We think these new analyses identify a number of interesting patterns. First in self-administration we show that the response to nicotine is present in the first sessions (FR1), and remains present throughout self-administration. Second, we show that the response to punishment dramatically overshadows that of nicotine initially, and this response to punishment declines as behaviour decreases . Also in punishment we show that the response to nicotine infusions is no longer significant above baseline in the first session. Third we show that the selective increase in activity prior to a response (i.e. active versus inactive) is only present in the first test session.3. In line with the previous comment, more details about the fibre photometry recording experiments would be helpful. Were the rats habituated to be tethered before to starting the procedure? State in the methods that the recording was performed every day of the task. Moreover, in the fibre photometry experiment the sessions of self-administration and punishment are reduced to 1h compared to the other experiments with 2-h/session. This difference suggests that the rats received less nicotine intake and context/cues exposure, it should be acknowledged. Did the authors compare the average nicotine intake between the experiments?We appreciate this comment, and have added information about this in the Methods in the revised manuscript.4. Concerning the representation of the calcium signal differences. To help the reader appreciate the variation of signal I would suggest representing the most relevant comparisons as a histogram of the average of the calcium transient .We have added Figure 2—figure supplement 2 showing the calcium transient calculated using mean df/f for the 2 second preceding the nose-poke and 5 seconds after. We do this for every data trace that we reported in the main figure (2).5. Although discussed in the paper one important limitation in the methodology and the interpretation of the results is the lack of consistency between the promoteor of the viral vector used. In the fibre photometry experiments, the authors used hSyn promoteor suggesting that they recorded the calcium signal from all neurons of the aIC (glutamatergic neurons and GABAergic interneurons) whereas in the chemogenetic experiments they used CaMKII promoteor allowing to specifically target the glutamatergic neurons. Fiber photometry recording using CaMKII promoteor would strengthen the results and the interpretation of the coding and causal properties of the aIC neurons.We appreciate this comment. In the revised discussion we raise this issue and discuss potential future directions related to these point6. The authors made an effort to include both male and female rats in their study. However, both sexes are not included in all the experiments making difficult to conclude about presence or lack of sex differences. It should be at least acknowledged.We have acknowledged this point in the revised manuscript.Reviewer #3 (Recommendations for the authors):This is an excellent paper and the authors should consider addressing the two points listed in the weaknesses section..1. The authors should consider discussing the limitations of interpretation of the fibre photometry data, particularly in regards to alternative interpretations of the increase in activity in the active nose poke -> punished condition and how this may be due to the multiple outcomes (nicotine infusion + punishment). It maybe useful to indicate on the graphs when the footshock and nicotine infusions took place.We thank the reviewer for this comment and have changed the figures to more clearly show the experimental design.2. The authors should consider testing if there is an effect on responding with chemogenetic inhibition of the anterior insular cortex in a procedure where responding is high (e.g. nicotine or food self-administration). Interpretation of the current data in Context B could be confounded by a floor effect.To address this potential issue, we conducted additional analysis on the latency to the first active and inactive nosepoke  which shows that there was no difference between inhibited aIC and control. In the discussion we argue that this observation further illustrates the specificity of the effect of aIC inhibition of nicotine seeking.


Multisystem Inflammatory Syndrome in Neonates (MIS-N) can occur following antenatal COVID- 19 infection in the mother. Here we report a rare case of a neonate with Hemophilia A and MIS-N.A 2-day-old baby presented with an intramuscular hematoma, neonatal seizures, and isolated activated partial thromboplastin time (APTT) prolongation. The neurosonogram showed a subdural hematoma. A diagnosis of Hemophilia A was made and was confirmed by factor 8 assay and genetic analysis. Supportive measures and Factor 8 replacement was initiated. A rising trend of inflammatory markers and an ongoing need for mechanical ventilation were noted. As there was a history of COVID-19 in the mother in the third trimester, MIS-N was diagnosed. The baby was treated with intravenous immunoglobulin (IVIG) and steroids, and there was an improvement in the clinical and laboratory markers. However, the baby developed seizures on day 16. There was an increase in the subdural hemorrhage and a further rise in inflammatory markers. A craniostomy and hematoma evacuation was done and the baby improved.The concurrent occurrence of hemophilia A with intracranial bleed, and MIS-N in a neonate is a diagnostic challenge. It is important to have a high index of suspicion to ensure timely diagnosis and treatment of MIS-N in this pandemic era. Hemophilia A is an X-linked coagulation disorder due to reduced levels of factor 8 with an incidence of 1 in 5000 male childbirth [In this report, we describe the occurrence of Multisystem Inflammatory Syndrome- Neonate (MIS-N) in a 2-day-old baby diagnosed with Hemophilia A. Transplacental transfer of antibodies occurs following maternal COVID-19 disease. This may lead to a hyperinflammatory state in the newborn called Multisystem Inflammatory Syndrome-Neonate (MIS-N). There have been very few cases of MIS-N reported so far [A 2-day-old male baby, born to a primigravida mother at 39 weeks of gestation by a forceps delivery, with a birth weight of 2.8 kg, presented to the Emergency Department (ED) with swelling on the left thigh, poor feeding, and lethargy. There was no family history of bleeding disorder. The baby was noted to have apnea in ED requiring intubation. After admission to the NICU, the baby was ventilated and stabilized. Examination showed bulging anterior fontanelle, anisocoria, and a 3 × 1 cm bluish swelling on the left thigh at the site of vitamin K injection. He had multiple episodes of seizures in the form of tonic posturing of all 4 limbs. Investigations showed anemia (Hemoglobin- 12.8 g/dL), normal leukocyte and platelet counts . C-reactive protein (CRP) was 13 mg/L . The coagulation profile showed normal prothrombin time (PT) and an isolated activated partial thromboplastin time (APTT) prolongation  -1.60, APTT>120S). A Neurosonogram on day 2 of life, showed a subdural hemorrhage (SDH) of 8 mm in the left frontotemporal region. Packed red cells and fresh frozen plasma were transfused after drawing samples for clotting factors. Factor 8 activity was < 1% and a diagnosis of hemophilia A was made. He was started on Factor 8 at 125 IU twice a day (targeting 100% factor levels), anticonvulsants, and antibiotics (Cefotaxime and Gentamicin). Computerized tomography (CT) scan on day 3 showed an SDH of 10 mm thickness in the left frontotemporoparietal region.On day 4 of life, the baby was hemodynamically stable and euglycemic but required continued mechanical ventilation and an increased oxygen requirement. There was also a rising trend in CRP (101 mg/L on day 4) despite changing the antibiotics to Meropenem and Amikacin  in the baby. Maternal and neonatal reverse transcription-polymerase chain reaction (RT-PCR) for SARS-CoV-2 were negative. Further investigations revealed elevated inflammatory markers: Lactate dehydrogenase (LDH) -1071 U/L, ferritin-448 ng/ml, and D-dimer-973 ng/ml. Hence the baby was given intravenous immunoglobulin (IVIG - 1 g/kg for 2 days) and methylprednisolone (1 mg/kg every 12 h for 6 days and tapered over 4 days).The baby improved and the ventilatory requirements were reduced. On day 6, the baby was extubated to room air. The CRP and other inflammatory markers also showed a decreasing trend. An echocardiogram (ECHO) showed prominent left coronaries with aneurysmal dilatation and normal ventricular function (ejection fraction − 77%). Factor 8 was continued targeting 100% levels. Factor 8 inhibitor assay was negative. The intracranial bleed was managed conservatively by the neurosurgery team.Serial monitoring of laboratory parameters showed an increase in CRP and other inflammatory markers on day 14  in a male baby with isolated APTT prolongation point to a coagulation disorder. Differential diagnoses to be considered are Factor 8,9,11 and 12 deficiency. In our case, low Factor 8 levels (severe deficiency < 1%) led to the diagnosis of Hemophilia A.More than one-third of patients with severe hemophilia are diagnosed in the first month of life . They coIn our case, we targeted 100% levels due to intracranial bleed. Despite appropriate treatment, clinical worsening was noted. A thorough investigation for the rare possibility of inhibitors was done and found to be negative. This prompted the treating team to consider an alternate or additional diagnosis. The temporal association of antenatal COVID − 19 infection in the third trimester, the presence of COVID antibodies in the baby, multisystem involvement, and elevated inflammatory markers suggested a diagnosis of MIS-N. Aneurysmal dilatation of proximal coronary arteries in the ECHO provided additional evidence in favor of the diagnosis.MIS-N is a newly reported entity with few cases reported as yet . The diaThis is the first report of MIS-N in a neonate with Hemophilia A to the best of our knowledge. Here we highlight the challenges faced in terms of diagnosis and treatment. Coagulation abnormalities may occur in Multisystem inflammatory syndrome due to COVID-19. Prolonged APTT, elevated fibrinogen, and D – dimer have been reported . HoweverNeuronal injury involving perihematomal neurons occurs in IC bleed. This triggers a cascade of events causing the elevation in inflammatory markers. However, there may be elevated inflammatory markers in both MIS-N and resolving or organizing IC bleed  , 9. TherAspirin or anticoagulants are recommended for the treatment of babies with dilated coronaries. However, given the ongoing IC bleed, we did not administer aspirin or anticoagulants.Many issues regarding the diagnosis and management of a neonate with MIS-N may arise especially when it occurs along with other diseases. This case highlights the need for a greater understanding of cases with the concurrent occurrence of MIS-N and other neonatal disorders.In this case, we report the occurrence of hemophilia A and COVID-associated MIS-N in a neonate. A high index of suspicion in the current pandemic period may help in diagnosing cases of MIS-N especially when it occurs concurrently with other neonatal disorders.